Enzymatic circuits for molecular sensors

ABSTRACT

In various embodiments a molecular circuit is disclosed. The circuit comprises a negative electrode, a positive electrode spaced apart from the negative electrode, and an enzyme molecule conductively attached to both the positive and negative electrodes to form a circuit having a conduction pathway through the enzyme. In various examples, the enzyme is a polymerase. The circuit may further comprise molecular arms used to wire the enzyme to the electrodes. In various embodiments, the circuit functions as a sensor, wherein electrical signals, such as changes to voltage, current, impedance, conductance, or resistance in the circuit, are measured as substrates interact with the enzyme.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/015,028 filed Jun. 21, 2018 and entitled “Enzymatic Circuits forMolecular Sensors.” The '028 application is a continuation of PCTApplication No. PCT/US18/29382, filed on Apr. 25, 2018 entitled“Enzymatic Circuits for Molecular Sensors,” which claims priority to andthe benefit of U.S. Provisional Patent Application Ser. No. 62/489,881filed Apr. 25, 2017 and entitled “Enzymatic Circuits for MolecularSensors,” the disclosures of which are incorporated herein by referencein their entirety.

FIELD

The present disclosure is generally directed to molecular sensors andmore particularly to molecular sensors in which an enzyme closes thecircuit between two electrodes.

BACKGROUND

The broad field of molecular electronics was introduced in the 1970's byAviram and Ratner. Molecular electronics achieves the ultimate scalingdown of electrical circuits by using single molecules as circuitcomponents. Molecular circuits comprising single molecule components canfunction diversely as switches, rectifiers, actuators and sensors,depending on the nature of the molecule. Of particular interest is theapplication of such circuits as sensors, where molecular interactionsprovide a basis for single molecule sensing. In particular, informativecurrent changes could include an increase, or decrease, a pulse, orother time variation in the current.

Notwithstanding the achievements in the field of molecular electronics,new molecular circuits that can function as molecular sensors are stillneeded. In particular, the need still exists for improved singlemolecule systems that can yield molecular information with greatersignal-to-noise ratios such that signals truly indicative of molecularinteractions are distinguishable from non-informative noise.

SUMMARY

In various embodiments, single molecule enzyme-based circuits aredisclosed wherein a single enzyme molecule is directly connected to apositive and negative electrode to form the circuit. These circuits arecapable of yielding highly informative signals of enzyme activity. Theseimproved signals have greater signal-to-noise levels such that thesignals are more distinguishable from noise, and these improved signalsinclude features that carry detailed information about the engagementbetween enzyme and the target substrate.

In various embodiments, a molecular sensor comprises an enzyme-basedmolecular circuit (conductive pathway) such as described herein. Such asensor having a polymerase enzyme is usable to sense sequenceinformation from a DNA template processed by the polymerase.

In various embodiments of the present disclosure, a molecular circuit isdisclosed. The circuit comprises: a positive electrode; a negativeelectrode spaced apart from the positive electrode; and an enzymeconnected to both the positive and negative electrodes to form aconductive pathway between the positive and negative electrodes.

In various aspects, the enzyme of the circuit may comprise a firstwiring point connected to the positive electrode and a second wiringpoint connected to the negative electrode.

In various aspects, the circuit may further comprise at least one armmolecule having first and second ends, the first end bonded to theenzyme and the second end bonded to at least one of the electrodes,wherein the at least one arm molecule acts as an electrical wire betweenthe enzyme and at least one of the electrodes.

In various aspects, the at least one arm molecule may be selected fromthe group consisting of a double stranded oligonucleotide, a peptidenucleic acid duplex, a peptide nucleic acid-DNA hybrid duplex, a proteinalpha-helix, a graphene-like nanoribbon, a natural polymer, a syntheticpolymer, and an antibody Fab domain.

In various aspects, at least one of the electrodes is connected to aninternal structural element of the enzyme.

In various aspects, the internal structural element may be selected fromthe group consisting of an alpha-helix, a beta-sheet, and a multiple ofsuch elements in series.

In various aspects, at least one of the electrodes may be connected tothe enzyme at a location of the enzyme capable of undergoing aconformational change.

In various aspects, at least one arm molecule may comprise a moleculehaving tension, twist or torsion dependent conductivity.

In various aspects, the enzyme may comprise a polymerase.

In various aspects, the polymerase comprises E. coli Pol I KlenowFragment.

In various aspects, the polymerase comprises a reverse transcriptase.

In various aspects, the polymerase comprises a genetically modifiedreverse transcriptase.

In various aspects, a molecular sensor comprises a circuit furthercomprising a positive electrode; a negative electrode spaced apart fromthe positive electrode; and a polymerase enzyme comprising E. coli Pol IKlenow Fragment connected to both the positive and negative electrodesto form a conductive pathway between the positive and negativeelectrodes, wherein the positive electrode and the negative electrodeeach connect to the polymerase at connection points within the majoralpha-helix of the polymerase extending between amino acids at position514 and 547.

In various aspects, a molecular sensor comprises a circuit furthercomprising a positive electrode; a negative electrode spaced apart fromthe positive electrode; and a polymerase enzyme connected to both thepositive and negative electrodes to form a conductive pathway betweenthe positive and negative electrodes, wherein the sensor is usable tosense sequence information from a DNA template processed by thepolymerase.

In various aspects, a molecular sensor comprises a circuit furthercomprising a positive electrode; a negative electrode spaced apart fromthe positive electrode; and a polymerase enzyme connected to both thepositive and negative electrodes to form a conductive pathway betweenthe positive and negative electrodes, wherein the positive electrode andthe negative electrode each connect to the polymerase at connectionpoints on the thumb and finger domains of the polymerase, and whereinsuch points undergo relative motion in excess of 1 nanometer as thepolymerase processes a DNA template.

In various aspects, the polymerase in this sensor is engineered to haveextended domains which produce a greater range of relative motion as thepolymerase processes a DNA template.

In various aspects, the polymerase in this sensor is engineered to haveadditional charge groups that variably influence the internal conductionpath as the enzyme processes a DNA template.

In various aspects, the polymerase in this circuit is a geneticallymodified form of E. coli. Pol I, Bst, Taq, Phi29, or T7 DNA polymerases,or a genetically modified reverse transcriptase.

In various aspects, a molecular circuit comprises: a positive electrode;a negative electrode spaced apart from the positive electrode; and anenzyme connected to both the positive and negative electrodes to form aconductive pathway between the positive and negative electrodes, whereinthe positive electrode and the negative electrode each connect to theenzyme at connection points in the enzyme comprising at least one of anative cysteine, a genetically engineered cysteine, a geneticallyengineered amino acid with a conjugation residue, or a geneticallyengineered peptide domain comprising a peptide that has a conjugationpartner.

In various aspects, this circuit further comprises a gate electrode.

In various embodiments, a method of sequencing a DNA molecule isdisclosed. The method comprises: providing a circuit further comprisinga positive electrode; a negative electrode spaced apart from thepositive electrode; and a polymerase enzyme connected to both thepositive and negative electrodes to form a conductive pathway betweenthe positive and negative electrodes; initiating at least one of avoltage or a current through the circuit; exposing the circuit to asolution containing primed single stranded DNA and/or dNTPs; andmeasuring electrical signals through the circuit as the polymeraseengages and extends a template, wherein the electrical signals areprocessed to identify features that provide information on theunderlying sequence of the DNA molecule processed by the polymerase.

In various embodiments, a method of molecular detection is disclosed.The method comprises, providing a circuit further comprising: a positiveelectrode; a negative electrode spaced apart from the positiveelectrode; a polymerase enzyme connected to both the positive andnegative electrodes to form a conductive pathway between the positiveand negative electrodes and a gate electrode; initiating at least one ofa voltage or a current through the circuit; exposing the circuit to atleast one of: a buffer of reduced ionic strength, a buffer comprisingmodified dNTPs, a buffer comprising altered divalent cationconcentrations, specific applied voltage on the primary electrodes, agate electrode voltage, or voltage spectroscopy or sweeping applied tothe primary electrodes or gate electrode; and measuring an electricalchange in the circuit.

BRIEF DESCRIPTION OF THE DRAWINGS

The subject matter of the present disclosure is particularly pointed outand distinctly claimed in the concluding portion of the specification. Amore complete understanding of the present disclosure, however, may bestbe obtained by referring to the detailed description and claims whenconsidered in connection with the drawing figures:

FIG. 1 illustrates the general concept of a molecular electroniccircuit;

FIG. 2 illustrates the general concept of engaging an enzyme to amolecular electronic circuit, such as to act as a sensor of enzymeactivity with its target;

FIG. 3 illustrates an enzyme that is wired directly into the currentpath, in accordance with various embodiments;

FIG. 4 illustrates an enzyme that is wired directly into the currentpath, with the connection made to an internal alpha-helix structurewithin the enzyme, in accordance with various embodiments;

FIG. 5 illustrates an enzyme that is wired directly into the currentpath, with the connection made to a series of two or more internalalpha-helix structures in series within the enzyme, in accordance withvarious embodiments;

FIG. 6 illustrates an enzyme that is wired directly into the currentpath, with the connection made to an internal beta-sheet structurewithin the enzyme, in accordance with various embodiments;

FIG. 7 illustrates an enzyme that is wired directly into the currentpath, such that connections are made to points of conformational changein the enzyme, to induce tension changes into the circuit during enzymeactivity;

FIG. 8 illustrates an enzyme that is wired directly into the currentpath, with additional connections made to stabilize the position of theenzyme;

FIG. 9 illustrates a schematic of an enzyme directly wired into thecurrent path of a circuit, in accordance with various embodiments,wherein the enzyme directly couples to the electrodes without the use ofarm molecules;

FIG. 10 illustrates a schematic of an enzyme directly wired by twopoints of contact into a circuit, as well as also having a one-pointconjugation to a molecular wire, utilizing one pair of electrodes tomeasure the combined conduction, in accordance with various embodiments;

FIG. 11 illustrates a schematic of an enzyme directly wired by twopoints of contact into a circuit, as well as also having a one-pointconjugation to a molecular wire, utilizing two pairs of electrodes tomeasure these two modes of conduction independently, in accordance withvarious embodiments;

FIG. 12 illustrates a protein structure view of the E. coli Pol I KlenowFragment Polymerase enzyme, illustrating the presence of alpha-helix,beta-sheet, and connecting loop structures;

FIG. 13 illustrates a schematic of a polymerase enzyme directly wiredinto the current path of a circuit, in accordance with variousembodiments, wherein a specific alpha-helix is used for the contacts,and molecular arms provide coupling to the electrodes;

FIG. 14 illustrates a schematic of a polymerase enzyme directly wiredinto the current path of a circuit, in accordance with variousembodiments, wherein a specific alpha-helix is used for the contacts,and the polymerase directly couples to the electrodes without the use ofarm molecules;

FIG. 15 illustrates a schematic of a polymerase enzyme directly wiredinto the current path of a circuit, in accordance with variousembodiments, wherein arms are wired to the points that undergo relativemotion when the finger and thumb domains change relative conformation;and

FIG. 16 illustrates a schematic of a polymerase enzyme directly wiredinto the current path of a circuit, and where additional connecting armsare wired to provide stabilization and fixed spatial orientation.

DETAILED DESCRIPTION

The detailed description of exemplary embodiments herein makes referenceto the accompanying drawings, which show exemplary embodiments by way ofillustration and their best mode. While these exemplary embodiments aredescribed in sufficient detail to enable those skilled in the art topractice the inventions detailed herein, it should be understood thatother embodiments may be realized and that logical, chemical, andmechanical changes may be made without departing from the spirit andscope of the inventions. Thus, the detailed description herein ispresented for purposes of illustration only and not of limitation. Forexample, unless otherwise noted, the steps recited in any of the methodor process descriptions may be executed in any order and are notnecessarily limited to the order presented. Furthermore, any referenceto singular includes plural embodiments, and any reference to more thanone component or step may include a singular embodiment or step. Also,any reference to attached, fixed, connected or the like may includepermanent, removable, temporary, partial, full and/or any other possibleattachment option. Additionally, any reference to without contact (orsimilar phrases) may also include reduced contact or minimal contact.

In various embodiments of the present disclosure, a molecular circuit isdisclosed. The molecular circuit comprises: a positive electrode; anegative electrode spaced apart from the positive electrode; and anenzyme connected to both the positive and negative electrodes to form aconductive pathway between the positive and negative electrodes. Invarious examples, the enzyme comprises a first wiring point connected tothe positive electrode and a second wiring point connected to thenegative electrode.

Definitions and Interpretations

As used herein, the term “enzyme” means a molecule that acts totransform another molecule, by engaging with a variety of substratemolecules. Such transformation could include chemical modification, orconformational modification. Common biological enzyme classes arepolymerases, ligases, nucleases, kinases, transferases, as well asgenetically modified forms of these molecules. Polymerases hereininclude reverse transcriptases and any genetically modified reversetranscriptase, capable of directly acting on an RNA template. Enzymesare most commonly proteins, but may be composed of multiple amino acidchains, and may also be complexed with other types of molecules, such asRNA in the case of the ribosome enzyme.

As used herein, the term “substrate” for an enzyme refers to any of themolecules that the enzyme specifically engages with in the course ofperforming a transformation. For example, in the specific case of a DNApolymerase, the substrate consists of both a template DNA and dNTPs. Inaddition to the substrates of the enzyme, the enzyme may also complexwith various co-factors that moderate its function or kinetics. Forexample, in the case of DNA polymerase, divalent cations such as Mg++are often essential cofactors, but not considered as substrates.

As used herein, the term “dNTP” or “dNTPs” refers to any of thedeoxynucleotide triphosphates involved in polymerase-based DNAsynthesis, or that can be engaged for such DNA synthesis, including bothnative and modified forms of such molecules.

As used herein, the term “buffer” for an enzyme refers to a solution inwhich the enzyme is viable and functional, and typically containing thesubstrates and co-factors needed for enzyme activity. Such an enzymebuffer may typically comprise salts, detergents, and surfactants, singlyor in various combinations, as well as specific cofactors, such asmagnesium or other divalent cations for a polymerase enzyme, along withthe substrates, such as DNA and dNTPs for a polymerase enzyme. Such abuffer herein may have its composition modified from standard forms,such as to enhance signal properties in a sensor exposed to the buffer.

As used herein, the term “electrode” means any structure that can act asan efficient source or sink of charge carriers. Most commonly thesewould be metal or semiconductor structures, such as those used inelectronic circuits. A pair of spaced apart electrodes herein maycomprise a source and drain electrode pair. In various embodiments ofthe present disclosure, a binding probe-based molecular circuit mayfurther comprise a gate electrode. When present, a gate electrode isused to apply a voltage rather than transfer charge carriers. Thus itsupports accumulation of charge carriers to produce a local electricfield, but is not intended to pass current. A gate electrode will beelectrically isolated from the primary conduction paths of the circuitby some form of insulating layer or material.

As used herein, the term “conjugation” means any of the wide variety ofmeans of physically attaching one molecule to another, or to a surfaceor particle. Such methods typically involve forming covalent ornon-covalent chemical bonds, but may also rely on protein-proteininteractions, protein-metal interactions, or chemical or physicaladsorption via intermolecular (Van der Waals) forces. There is a largevariety of such methods know to those skilled in the art of conjugationchemistry. Common conjugation methods relevant to preferred embodimentsherein include thiol-metal bonds, maleimide-cysteine bonds, materialbinding peptides such as gold binding peptides, and click chemistries.

As used herein, the term “initiating,” in the context of an electricalparameter, is intended to be broader than the concept of “applying” anelectrical value. For example, an electrical current may be initiated ina circuit. Such initiating of a current may be the result of applying avoltage to the circuit, but may be from other actions to the circuitbesides applying a voltage. Further, a voltage may be initiated in acircuit. Such initiating of a voltage may be the result of applying acurrent to the circuit, but may be from other actions to the circuitbesides applying an electrical current. In other examples, a voltage ora current may be initiated in one portion of a circuit as the result ofapplying a voltage or a current to the overall circuit. In anon-limiting example, a flow of electrons initiated from a negative to apositive electrode in a circuit of the present disclosure may becontrolled by the voltage applied to the gate electrode of the circuit.

In various embodiments of the present disclosure, a molecular sensorcomprises an enzyme connected to both a positive and a negativeelectrode to complete a circuit. Interactions of the enzyme with varioussubstrates are detectable as changes in the current or other electricalparameter measured across the circuit. The present molecular sensordiffers from the general concept of a molecular electronic circuit inthat the enzyme is directly “wired” to both the positive and negativeelectrodes rather than bonded to a molecular bridge molecule that spansthe gap between the electrodes to complete a circuit.

In various aspects of the disclosure, at least one of a voltage or acurrent is initiated in an enzyme-based molecular circuit. When a targetinteracts with the enzyme, electrical changes in the circuit are sensed.These electrical changes, or informative electrical signals, may includecurrent, voltage, impedance, conductivity, resistance, capacitance, orthe like. In some examples, a voltage is initiated in the circuit andthen changes in the current through the circuit are measured assubstrates interact with the enzyme. In other examples, a current isinitiated in the circuit, and changes to voltage in the circuit aremeasured as substrates interact with the enzyme. In other examples,impedance, conductivity, or resistance is measured. In examples whereinthe circuit further comprises a gate electrode, such as positionedunderneath the gap between the positive and negative electrodes, atleast one of a voltage or current may be applied to the gate electrode,and voltage, current, impedance, conductivity, resistance, or otherelectrical change in the circuit may be measured as substrates interactwith the enzyme.

FIG. 1 illustrates the general concept of a molecular electronic circuithaving a bridge molecule 10 attached to and bridging the gap 12 betweenelectrodes 15, as well as some type of conjugation group 20 or othermechanism that binds the molecule to the electrodes (depicted as smallshaded squares). FIG. 1 further illustrates that a current, (i), maypass through this molecule and be measured versus time, (t), as shown inthe inset plot 25.

FIG. 2 illustrates a molecular electronic sensor in which an enzyme 30is conjugated to the molecular bridge component 31 spanning theelectrodes 32, wherein monitoring the current provides a sensor for theenzyme engaging with and processing its target substrate 35 when exposedto a suitable buffer solution. In such a sensor system, the local chargeperturbations that result from the target substrate engaging with theenzyme perturb charge transport through the primary bridge component,and are thus registered as a change in conductivity or current versustime, as indicated by the step-up change in the current (i) vs. time (t)current plot inset 38 in FIG. 2.

In contrast to the general molecular circuit concept as depicted inFIGS. 1 and 2, in various embodiments of the present disclosure amolecular sensor comprises a single enzyme molecule directly wired intothe circuit path, such that all electrical current passing through themolecular circuit must flow through the enzyme. Thus the enzyme is anessential conduction path in the circuit, like an electronic componenton a circuit board. The present concept is illustrated generally in FIG.3, which shows an enzyme 42 connected between molecular arms 40. Byforcing all current in the circuit to pass through the enzyme, thecurrent carriers are forced to pass closer to the precise location ofelectrochemical interactions between the enzyme and target substrate 45,thereby causing such interactions to have greater impact on the currentcarriers, and, in turn making the overall current more sensitive to thedetails of these interactions. This is illustrated schematically by thecurrent versus time, (i vs. t), plot inset 50 in FIG. 3, wherein thecurrent step is shown to be much larger than that produced by theconfiguration of FIG. 2, and also includes additional features notpresent in a current versus time plot such as depicted in FIG. 2. Thehigher current step provides improved signaling. Related methods andpreferred embodiments herein promote improved signaling of enzyme-basedmolecular sensors. Further, the configuration of the enzyme as anessential conduction path is fundamentally different from the commonconfiguration of FIG. 2, in which there are many conduction paths thatdo not pass through the enzyme, and where potentially none of the chargecarriers actually traverse the enzyme, and where there is no meansprovided to direct charge carriers to pass near key active sites withinthe enzyme.

In various embodiments, the enzyme may be coupled to both positive andnegative electrodes at two or more points, such as to ensure that chargecarriers traversing the molecular structure pass into and out of theenzyme.

As shown in the embodiment of FIG. 3, two molecular arms are conjugatedto the enzyme to provide physical anchors and entry and exit paths forthe current through the enzyme. Such arms may comprise any convenientmolecule that provides a conducting connection or semi-conductingconnection between the enzyme and the electrodes. Further, moleculararms may provide spanning length extensions, to help span a largerelectrode gap 55 that is wider than the 3D structure of the enzyme. Sucharms may also provide the advantage of keeping the enzyme away fromcontacting the electrodes 65 where unfavorable or damaging interactionsmay occur with the electrodes, such as a denaturing adsorption to theelectrode. Such arms may also provide for more compatible or efficientcoupling to the electrodes, such as by coupling to the electrodes viachemical groups that are not readily found or made available on theenzyme. For example, in one specific embodiment, the electrode comprisesgold and the molecular arm includes a thiol group, such that the armcouples to the gold electrode via well-known thiol-gold binding. Thusthe molecular arm accomplishes the binding while the enzyme may not havesuch available thiol groups. Or, in another embodiment, the arms maypresent a click-chemistry binding group, for coupling to electrodes thatare derivatized with the cognate binding partners for the clickchemistry.

In various embodiments, molecular arms comprise some form of conjugation60 to the enzyme, as well as their conjugations or couplings to theelectrodes. Many conjugation chemistries can be employed for thispurpose. In a non-limiting example, such conjugation comprises chemicalcrosslinking, which can preferentially couple suitable chemical groupson the arms to amino acid residues on the enzyme. In variousembodiments, a maleimide group on the arm couples to a surface cysteineon the enzyme. In other aspects, genetically modified versions of anenzyme may be created and employed, such as enzymes comprising specificamino acids or protein domains engineered into their amino acidstructure that provide specific conjugation sites. For example, cysteineamino acids engineered at specific sites on the enzyme provide for theattachment point of arms that present a maleimide group. Two suchcysteine sites conjugate to two maleimide derivatized arms to produce aconfiguration such as that shown in FIG. 3. In this case, one or morenative cysteines that would provide competing arm binding sites may be“engineered out” of the amino acid sequence. If not all such sites canbe removed, it is possible to use various purification methods fromsynthetic chemistry to isolate desired enzyme-arm conjugates fromunwanted configurations. In other variations, genetic methods are usedto engineer into the amino acid sequence of the enzyme amino acidscomprising residues that uniquely conjugate to a cognate group on thearms. This variation includes cases where non-standard amino acids areemployed, such as amino acids modified to present a click-chemistrygroup, via protein expression systems that use a modified genetic codeand modified transfer RNAs to put non-native amino acids at specificsequence sites in an expressed enzyme protein.

In other embodiments, a peptide domain that specifically binds with acognate group on the arms is engineered into the sequence of a proteinenzyme. In one such embodiment, a peptide that is an antigen to anantibody is engineered into the enzyme, and the Fab binding domain ofthe antibody is used on the arms. One such embodiment is to use the FLAGpeptide motif DYKDD, and any suitable ANTI-FLAG Fab domain. Any otherpeptide antigens and their cognate Fab domains can similarly be used toconjugate arms to specific sites in an engineered enzyme protein, byengineering the peptide antigen into the desired conjugation sites onthe enzyme. Other such peptide domains make use of theSPY-TAG/SPY-CATCHER protein-protein binding system, by engineeringeither the SPY-TAG domain or the SPY-CATCHER domain into the enzymeprotein, and placing the cognate domain in the arms. When engineeringsuch peptide binding domains into the enzyme, another embodiment is toadd short linker peptide sequences flanking the target peptide, toenhance the availability of the domain for binding. Such short linkersmay comprise short glycine and serine rich linkers, as are known tothose skilled in the art of protein engineering, including, but notlimited to, the linker amino acid sequences G, GS, GSG, GGSG, etc.

In various examples, the arm molecules comprise any molecules thatprovide for conduction of charge carriers into and out of the enzyme. Incertain embodiments, such arms comprise molecular wires from the manyforms known in field of molecular electronics, functionalized withsuitable conjugation and binding groups for wiring to electrodes andenzyme. In various aspects, such arms may comprise single stranded DNA,double stranded DNA, peptides, peptide alpha-helices, antibodies, Fabdomains of antibodies, carbon nanotubes, graphene nanoribbons, naturalpolymers, synthetic polymers, other organic molecules with p-orbitalsfor electron delocalization, or metal or semiconductor nanorods ornanoparticles. In further embodiments, the arms may comprise doublestranded DNA with thiol-bearing groups at one end, and maleimide at theother end that couples to the enzyme, or a peptide alpha-helix with acysteine or gold binding peptide at one termini, and a maleimide at theother end that couples to the enzyme, or a graphene nanoribbon withthiol-bearing groups at one end, and a maleimide bearing group at theother end that couples to the enzyme. In certain embodiments, the twoarm molecules used to couple an enzyme to two electrodes are identicalmolecules, and in other embodiments, the two arm molecules are differentmolecules. In some examples, there may be a “positive electrode” arm anda “negative electrode” arm, providing for oriented binding of an enzymeto the corresponding “positive” and “negative” electrodes in FIG. 3.

In various embodiments, arm conjugation points connect directly tospecific protein structural elements within the enzyme. A non-limitingexample is illustrated in FIG. 4, where the arms 75 are shown wireddirectly to an alpha-helix structure 70 in the enzyme 72. Suchstructural elements provide preferential conduction paths through theenzyme. Direct wiring to natural conduction paths in the enzyme guidecurrent closer to active regions of interest within the enzyme, such assubstrate binding pockets, and may thereby provide for further enhancedcurrent signals, or current signals that carry more information onenzyme-substrate interactions. For example, one embodiment is shown inFIG. 4, where the arms wire directly to an alpha-helix that spansbetween two points on or near the surface of the enzyme. Another exampleis shown in FIG. 5, where the arms 80 wire directly to two alpha-helices(the first alpha-helix 85 and the second alpha helix 87) that appear inseries internally in the enzyme, with a single connecting loop 90separating them. Yet another embodiment is shown in FIG. 6, where thearms 95 wire directly to two points 98 on a beta-sheet 100 internal tothe enzyme 102.

In general, a protein enzyme will have a 3D structure that includes wellknown secondary structural elements such as alpha-helices andbeta-sheets. These are primarily hydrogen bonded structures that canprovide discrete conduction paths through the body of the enzyme, to theextent that current carriers, such as electrons, may efficiently hopalong such structures, or along the hydrogen bonds that define suchstructures, with less resistance than otherwise hopping or tunneling offsuch structures. These structures provide preferential conduction pathsthat will channel charge carriers, and by selecting such structures,charge is forced to pass close to active regions of the enzyme, andcurrent-based sensing of the activity will be improved. Having the armsdirectly connected to such structures, or within a small number of aminoacids of the termini of such structures, the current flowing along thesedesirable paths is maximized, and thus the desirable signals that comefrom the current along such paths is maximized. In this way, currentgoing elsewhere within the enzyme is minimized, and thus the noise fromprobing these less informative regions is also minimized.

In various examples, the wiring can be to such structures that appear inthe enzyme “in series”, such as for example, two alpha-helices in seriesas indicated in FIG. 5, or a beta-sheet in series with an alpha-helix,or three successive alpha-helices. In general, each successive elementin series appears in the enzyme primary amino acid sequence as separatedfrom the previous element by a small number of amino acids, such 0, 1,2, or up to approximately 10 amino acids, which typically form aconnecting loop in the secondary structure. Wiring of elements in seriesmay also be achieved by wiring to structures that are not contiguous inthe primary amino acid sequence of the enzyme, but are nonethelessspatially contiguous and in conductive contact, and form a preferredconduction path, owing to hydrogen bonding, salt bridges, disulfidebridges, or other types of molecular interaction that establishsecondary, tertiary or quaternary protein structure and that can providea clearly defined and favorable conduction path from one structuralelement (beta-sheet, alpha-helix) to another. These structural elementsof interest for wiring, either in isolation or in series, are mostevident when examining the 3D structure of the proteins involved, as canbe observed from the crystal structures, and in particular, byexamination of the protein structures obtained by X-ray or NMRcrystallography. This useful form of structural information isillustrated by the polymerase enzyme structure shown in FIG. 12.

In other embodiments, the arms are wired to points on the enzyme thatundergo conformation changes or relative motion during enzyme function,such as illustrated in FIG. 7. In this case, the arms 105 are wired totwo points 110 that are indicated as having relative motion, asillustrated in the inset 108, during enzyme activity. This configurationcan enhance signaling by several means. First, such motions can changethe tension in the arms, and it is known that tension changes inmolecules can change their conductivity, thus the motion may betransduced via tension into a change in conductivity of the arms, whichconsequently show up in the current signals. In this way, the currentmay contain information about the conformational changes in the enzyme.Second, similarly, this configuration can cause tension in the enzyme asit changes conformation, and thus alter conductivity of the enzyme.Since the enzyme is an essential current path, the conformation changeswould transduce into current changes, and thereby represent conformationinformation in the sensing current. This configuration could alsoenhance signaling by altering the conformational changes of the enzyme,which may in some situations lead to an enhanced signal, for either anative enzyme, or one engineered to specifically benefit from suchconformation-sensitive wiring. In one embodiment, an enzyme isengineered to have extended regions that undergo greater conformationalchange or relative motion (e.g. as demonstrated by extending the lengthof the two tips of the scissor-shaped enzyme indicated in FIG. 7), so asto enhance the range of motion, and therefore the range of tensionchanges in the arms and enzymes.

In other aspects, conformational changes in the enzyme, such as wheninduced binding occurs between the enzyme and a substrate, aretranslated into a twist, torque or rotation of at least one arm, andthat twist, torsion or rotation alters the conductivity of the arm. Onesuch example is an arm comprising an organic polymer further comprisingpolycyclic aromatic rings, such as polythiophene or polyphenylene,whereby previously lined up p-orbitals are rotated out of alignment byC—C bond rotation when the arm is twisted, torqued or rotated inresponse to an enzyme conformational change. When the arm is twisted,torqued or rotated, the electrons have impeded delocalization throughthe organic polymer. In certain embodiments, such impeded flow may acton only a subset of the charge carriers, depending on, for example, thepolarization or other quantum state of the charge carrier, such as spinpolarization of an electron charge carrier, or the momentum or energystate of the charge carrier.

Another example is illustrated in FIG. 8, wherein the molecular sensorcircuit comprises more than two arms, for example, 3 arms (the first arm115, the second arm 116, and the third arm 117). The benefits to usingadditional enzyme wiring points and associated arms include addition ofother desirable conduction paths through the enzyme, and increasing theoverall conduction through the enzyme. Such additional arms may alsoprovide stabilization, impose a spatial orientation (such as to orientan active site), or otherwise reduce physical degrees of freedom orconformational entropy, which may improve sensing by reducing thevariability in conduction that comes from the system having moreaccessible conformations. Such additional arms may be conductive, butthey can also be insulating if they are present primarily to providestability, orientation, or reduction in spatial degrees of freedom. Suchadditional arms may connect to the electrode, or to other portions ofthe structure, such as to a substrate supporting the electrodes. Sucharms may connect to additional electrodes in a system comprising morethan two electrodes, including the case of a system with a gateelectrode, such as a buried gate electrode. Connection to a gateelectrode may refer to connection to the conductive portion of the gate,or connection to the insulating layer that separates actual conductivegate from the circuit, or, in the case of a buried gate, the surfacelayer above the buried gate, such as the connection to the surfaceillustrated in FIG. 16.

As illustrated in FIG. 9, the enzyme may be connected to the electrodesdirectly 120, as an essential conduction path, without the use of armmolecules. In this case, groups on the enzyme directly couple to theelectrodes. Or, in another embodiment, one wiring connection comprisesdirect coupling to the enzyme, the other via an arm molecule. Theadvantages of this arm-less configuration include minimizing the lengthof the conduction path, since the parts of the conduction path outsideof the enzyme can be sources of unwanted noise, resistance orcapacitance. The considerations above for the case of wiring with armsgenerally also apply to the special case of an arm-less configuration aswell as the configuration of a single arm combined with direct enzymecoupling. Specifically, in embodiments lacking arms, the enzyme maystill be wired via internal structures, or at points of conformationalchange.

A sensor comprising a directly wired enzyme as an essential conductionpath may have its signal performance enhanced through variousenvironmental factors. For example, the choice of buffer, bufferadditives, temperature and applied voltage may be modulated to improvethe signal quality. In particular, since enzymes may complex withvarious cofactors that modulate their kinetics, and the salt levels inthe buffer also impact enzyme kinetics, as does temperature, thesefactors may be used to improve signaling performance. In addition, theoverall ionic strength of the buffer solution defines the Debye lengthin the solution, that is the distance over which electric fields extendin solution, and can impact the extent to which current carriers passingthrough the enzyme are influenced by the charge distributions of theenzyme and substrate, and thus buffer ionic strength or total saltconcentration is another means of influencing or enhancing thesignaling. In embodiments utilizing a polymerase enzyme, the divalentcation content of the buffer is known to influence enzyme activity, andthe choice of divalent cation, for example from among Mg++, Mn++, Ni++,Zn++, Co++, Ca++, Cr++, and their concentration may be optimized toimprove the signaling from a polymerase wired as an essential conductionpath.

The applied driving voltage may be optimized to improve the signalingfrom an enzyme wired as an essential conduction path. Based on energybarriers within the enzyme, certain voltages may lead to improvedsignaling performance. In addition to an applied voltage, variousembodiments may also have a gate electrode, such as a buried gate belowthe lower substrate indicated in FIG. 3, such that voltages applied tothe gate electrode further modulate the signaling properties of theenzyme circuit. Certain embodiments may employ voltage spectroscopy,wherein the driving or gate voltages are swept through a range ofvalues, and the signal of interest is in the response from this sweep,which contains information on the interaction between the enzyme and itssubstrates.

In general, the molecular circuit sensors of the present disclosurecomprise the wiring of an enzyme with at least two points of electricalcontact, so as to make the enzyme an essential conduction path, incontrast to the configuration of FIG. 2. Two-point wiring of the enzymemay be combined with a conjugation 125 to one or more molecular arms127, as shown in FIGS. 10 and 11. In these embodiments, the current canbe both driven through the enzyme, for sensing, and the enzyme can alsomodulate current through the other molecular wire, as an additionalsensing mode. In FIG. 10, these conduction modes are monitored by asingle electrode pair 130, and combine to produce a single current,whereas in FIG. 11, these two conduction modes can be monitored by twoseparate electrode pairs (a first electrode pair 135 and a secondelectrode pair 140), producing two current measurements 145. In certainembodiments, the sensor may comprise an enzyme 126 wired up with two ormore points of contact as a conduction path, in conjunction withadditional sensor configuration features. Wiring the enzyme at twopoints, with input and output electrical contacts, can provide enhancedsignaling. Other possible and non-limiting configurations areillustrated in FIGS. 10 and 11.

In various embodiments, a molecular circuit sensor comprises apolymerase enzyme. FIG. 12 shows a representative polymerase enzyme 150,the Klenow Fragment of E. coli DNA Polymerase I. FIG. 12 illustrates aribbon diagram of the enzyme structure, from two different views, withthe enzyme engaged with a double-stranded DNA template 170. The enzymeprimary structure is a single amino acid sequence of 605 amino acids.The secondary structure and tertiary structure of the enzyme aredepicted in FIG. 12. This figure shows the structural elements of theenzyme. In particular, there are 28 distinct alpha-helix elements 160,and two major beta-sheet elements 165, separated by short flexible loopsegments 155. Such structural elements are similarly present in othertypes of polymerases and other enzyme proteins in general. In the courseof enzyme activity, these structural features engage in electrical,chemical, mechanical and conformational perturbations, and wiring tothese features within an electrical circuit can transduce theseperturbations into measured signals.

FIG. 13 shows an embodiment where the polymerase 177 is wired as anessential conduction path, and specifically wired to the ends of a longalpha-helix 180 that passes through the center of the enzyme. Thisalpha-helix was chosen because it passes very near to the active pocketof the polymerase, and therefore can provide for enhanced currentsensing of the polymerase activity as it binds to a primed strand andextends the primer through incorporation of dNTPs. Other alpha-helicesin the structure will provide other sensing opportunities, and otherembodiments comprise wiring to such alpha-helix structures, or thebeta-sheet structures, or other such structures occurring in series. Thearms 175 indicated in FIG. 13 may comprise double strandedoligonucleotides terminated with a maleimide, which couples to acysteine genetically engineered into a precise location in a mutant formof the polymerase. In another embodiment, the arms comprise a proteinalpha-helix terminated with a maleimide which couples such a cysteine.One specific embodiment of such a mutant polymerase includes cysteine(C) placed at the conjugation points indicated in FIG. 3, which arisefrom replacing the glutamine (Q) at amino acid position 548 by C, andreplacing the serine (S) at amino acid position 508 by C, and these twolocations lie just outside and bracket a single long (37 amino acid)alpha-helix that extends from amino acid position 514 to 547. In certainembodiments, the mutant polymerase further has the single native C atamino acid position 584 replaced by a non-cysteine, such as S, so as toprovide exactly the two sites for coupling of the maleimide terminatedarms, via the well-known maleimide-cysteine conjugation. Making suchamino acid substitutions to introduce cysteines should be done in amanner that does not alter highly conserved amino acids (as conserved incomparison to other polymerases), does not alter amino acids in thealpha-helix or other structural elements that are the target of thewiring, does not alter amino acids directly participating in criticalenzyme function, such as those that interact directly with the structureof the substrate binding pocket, the DNA substrate or the dNTPsubstrates. Similar selection principles apply to other enzymes as wellwhen mutating in cysteine as a maleimide conjugation point.

FIG. 14 illustrates an alternative embodiment where the mutantpolymerase 177 of FIG. 13 is directly conjugated 185 to the electrodes187, coupling to the internal alpha-helix, without the use of connectingarms. This coupling can be achieved, for example, by utilizing goldelectrodes, and a gold-binding peptide (GBP) with a maleimide terminus,such that the maleimide conjugates the GBP to the mutant polymerase atthe cysteine sites described above, and the GBP conjugates to the goldelectrode, thereby wiring in the polymerase via these two cysteinesites. Other embodiments of direct maleimide-mediated conjugations tothe electrodes are enabled by using conjugating groups having the formX-maleimide bonded to the cysteines on the polymerase, such that X is agroup that then binds to the electrode surface.

FIG. 15 illustrates an alternative embodiment, where the mutantpolymerase 177 of FIG. 13 is wired to the electrodes 212 usingconnecting arms 210 through an alpha-helix 200 adjacent to the bindingcleft 205 of the polymerase. The connecting arms are wired to two points215 on the polymerase. In embodiments, these two points have the abilityto move relative to each other, thereby allowing for changes inconductivity and enhanced signaling.

FIG. 16 illustrates an embodiment in which multiple arms 190 are used towire up the polymerase 195 as an essential conducting path, as well asto stabilize its position and orientation relative to the electrodes andsubstrate. The lower pair of arms indicated can be either conducting orinsulating, in accordance with various embodiments.

In various embodiments, a circuit comprises an enzyme wired in as anessential conduction path. The circuit may comprise first and secondwiring points, connecting to a first and a second electrode such as apositive electrode and a negative electrode.

In various embodiments, the circuit may further comprise at least onearm molecule having two ends, one end bonded to the enzyme and the otherend bonded to at least one of the electrodes, wherein the at least onearm molecule acts as an electrical wire between the enzyme molecule andat least one of the electrodes. Such an arm molecule may be selectedfrom the group consisting of a double stranded oligonucleotide, apeptide nucleic acid (PNA) duplex, a PNA-DNA hybrid duplex, a proteinalpha-helix, a graphene-like nanoribbon, a natural polymer, a syntheticorganic molecule e.g. a synthetic polymer, and an antibody Fab domain.In other examples, the enzyme is wired directly to the electrodeswithout the use of any arm molecules. The wiring may be to an internalstructural element in the enzyme, such as an alpha-helix, or a betasheet, or multiple such elements in series.

In various embodiments, a circuit comprises an enzyme wired at pointsthat undergo relative conformational change. In certain aspects, armscomprise molecules that have a tension dependent conductivity. In otherexamples, arm molecules may have torsion or twist dependentconductivity. Additional wiring points may be used to couple the enzymeat additional sites.

In various embodiments, a circuit comprises a polymerase enzyme, such asfor example, E. coli Pol I Klenow Fragment, wherein the wiring is to themajor alpha-helix extending between amino acids at position 514 and 547.Such connection may rely on the placement of genetically engineeredcysteines at or near these amino acid positions. Circuits comprising apolymerase may be used to sense sequence information from a DNA templateprocessed by the polymerase.

A circuit in accordance to various embodiments of the present disclosuremay be exposed to a solution containing primed single stranded DNA,and/or dNTPs, wherein the current through the circuit is measured as thepolymerase engages and extends a template, and the resulting signals areprocessed to identify features that provide information on theunderlying sequence of the DNA molecule processed by the polymerase.

The connection between the enzyme molecule and at least one of thepositive electrode and negative electrode may comprise any one of: anative cysteine, a genetically engineered cysteine, a geneticallyengineered amino acid with a conjugation residue, or a geneticallyengineered peptide domain comprising a peptide that has a conjugationpartner. In certain aspects, the wiring is to points on the thumb andfinger domain of the enzyme, where such points undergo relative motionin excess of 1 nm as the polymerase processes a DNA template. In otheraspects, the polymerase is engineered to have extended domains thatproduce a greater range of relative motion as the polymerase processes aDNA template. For example, conformational changes in an enzyme may beaccentuated by extending various domains in the enzyme. A polymeraseenzyme may also be engineered to have additional charge groups thatvariably influence the internal conduction path as the enzyme processesa DNA template.

In various embodiments, a circuit is exposed to a solution comprisingmodified dNTPs that variably influence the internal conduction path asthe enzyme processes a DNA or RNA template. In some cases, thepolymerase enzyme is a genetically modified form of one of: E. coli. PolI polymerase, Bst polymerase, Taq polymerase, Phi29 polymerase, T7polymerase, and reverse transcriptase. In other examples, a circuit isexposed to one or more of the conditions of: a buffer of reduced ionicstrength, a buffer comprising modified dNTPs, a buffer comprisingaltered divalent cation concentrations, specific applied voltage on theprimary electrodes, a gate electrode voltage, or voltage spectroscopy orsweeping applied to the primary electrodes or gate electrode.

In various embodiments, the polymerase enzyme comprises a reversetranscriptase or genetically modified reverse transcriptase, capable ofdirectly acting on an RNA template. Use of a reverse transcriptase inthese circuits has the benefit that the reverse transcriptase candirectly process an RNA template, and therefore provide a means fordirectly sequencing RNA molecules. In various aspects, this reversetranscriptase could be any monomeric reverse transcriptase or agenetically modified form thereof, such as Moloney murine leukemia virusreverse transcriptase, porcine endogenous retrovirus reversetranscriptase, bovine leukemia virus reverse transcriptase, mousemammary tumor virus reverse transcriptase, or a heterodimeric reversetranscriptase such as human immunodeficiency virus reverse transcriptaseor Rous sarcoma virus reverse transcriptase.

In certain examples, a method of sequencing a DNA molecule is disclosed.The method comprises: providing an enzyme-based molecular circuit havingspaced-apart positive and negative electrodes and a polymerase enzymemolecule connected to both the positive and negative electrodes to forma conductive pathway between the electrodes; initiating at least one ofa voltage or a current through the circuit; exposing the circuit to asolution containing primed single stranded DNA and/or dNTPs; andmeasuring electrical signals through the circuit as the polymeraseengages and extends a template, wherein the electrical signals areprocessed to identify features that provide information on theunderlying sequence of the DNA molecule processed by the polymerase.

In other aspects, a method of molecular detection is disclosed. Themethod comprises: (a) providing an enzyme-based molecular circuit havingspaced-apart positive and negative electrodes, a polymerase enzymemolecule connected to both the positive and negative electrodes to forma conductive pathway between the electrodes, and a gate electrode; (b)initiating at least one of a voltage or a current through the circuit;(c) exposing the circuit to at least one of: a buffer of reduced ionicstrength, a buffer comprising modified dNTPs, a buffer comprisingaltered divalent cation concentrations, specific applied voltage on theprimary electrodes, a gate electrode voltage, or voltage spectroscopy orsweeping applied to the primary electrodes or gate electrode; and (d)measuring an electrical change in the circuit.

Enzyme-based molecular sensors and methods of making and using same areprovided. References to “various embodiments”, “one embodiment”, “anembodiment”, “an example embodiment”, etc., indicate that the embodimentdescribed may include a particular feature, structure, orcharacteristic, but every embodiment may not necessarily include theparticular feature, structure, or characteristic. Moreover, such phrasesare not necessarily referring to the same embodiment. Further, when aparticular feature, structure, or characteristic is described inconnection with an embodiment, it is submitted that it is within theknowledge of one skilled in the art to affect such feature, structure,or characteristic in connection with other embodiments whether or notexplicitly described. After reading the description, it will be apparentto one skilled in the relevant art(s) how to implement the disclosure inalternative embodiments.

Benefits, other advantages, and solutions to problems have beendescribed with regard to specific embodiments. However, the benefits,advantages, solutions to problems, and any elements that may cause anybenefit, advantage, or solution to occur or become more pronounced arenot to be construed as critical, required, or essential features orelements of the disclosure. The scope of the disclosure is accordinglyto be limited by nothing other than the appended claims, in whichreference to an element in the singular is not intended to mean “one andonly one” unless explicitly so stated, but rather “one or more.”Moreover, where a phrase similar to ‘at least one of A, B, and C’ or ‘atleast one of A, B, or C’ is used in the claims or specification, it isintended that the phrase be interpreted to mean that A alone may bepresent in an embodiment, B alone may be present in an embodiment, Calone may be present in an embodiment, or that any combination of theelements A, B and C may be present in a single embodiment; for example,A and B, A and C, B and C, or A and B and C.

All structural, chemical, and functional equivalents to the elements ofthe above-described various embodiments that are known to those ofordinary skill in the art are expressly incorporated herein by referenceand are intended to be encompassed by the present claims. Moreover, itis not necessary for a device or method to address each and everyproblem sought to be solved by the present disclosure, for it to beencompassed by the present claims. Furthermore, no element, component,or method step in the present disclosure is intended to be dedicated tothe public regardless of whether the element, component, or method stepis explicitly recited in the claims. No claim element is intended toinvoke 35 U.S.C. 112(f) unless the element is expressly recited usingthe phrase “means for.” As used herein, the terms “comprises”,“comprising”, or any other variation thereof, are intended to cover anon-exclusive inclusion, such that a molecule, composition, process,method, or device that comprises a list of elements does not includeonly those elements but may include other elements not expressly listedor inherent to such molecules, compositions, processes, methods, ordevices.

1. A circuit comprising: a first electrode; a second electrode spacedapart from the first electrode; a first arm molecule having a first endand a second end, the second end of the first arm molecule electricallycoupled to the first electrode; a second arm molecule having a first endand a second end, the second end of the second arm molecule electricallycoupled to the second electrode; and an enzyme, wherein the first end ofeach of the first and second arm molecules are conjugated to twodistinct sites on the enzyme so that a portion of the enzyme between thetwo distinct sites is included in the circuit.
 2. The circuit of claim1, wherein the first electrode is a positive electrode and the secondelectrode is a negative electrode, or wherein the first electrode is anegative electrode and the second electrode is a positive electrode. 3.The circuit of claim 1, further comprising an additional electrodeconfigured as a gate or buried gate electrode.
 4. The circuit of claim1, wherein the portion of the enzyme included in the circuit comprisesan alpha-helix or a beta-sheet.
 5. The circuit of claim 4, wherein thealpha-helix or the beta-sheet portion of the enzyme provide a conductionpathway close to an active region of the enzyme.
 6. The circuit of claim1, wherein the conjugation between the first end of each of the firstand second arm molecules and the two distinct sites on the enzymecomprise maleimide-cysteine conjugations.
 7. The circuit of claim 1,wherein the electrical coupling between the second end of each of thefirst and second arm molecules and the first and second electrodescomprise thiol-gold coupling.
 8. The circuit of claim 1, wherein atleast one of the first and second arm molecules comprises a moleculehaving tension, twist, or torsion dependent conductivity.
 9. The circuitof claim 1, wherein the first and second arm molecules independentlycomprise a single-stranded DNA oligonucleotide, a double-stranded DNAoligonucleotide, a peptide nucleic acid (PNA) duplex, a PNA-DNA hybridduplex, a polypeptide, an antibody, an antibody Fab domain, a carbonnanotube, or a graphene nanoribbon.
 10. The circuit of claim 1, whereinthe enzyme comprises a polymerase, a ligase, a nuclease, a kinase, atransferase, or a reverse transcriptase.
 11. The circuit of claim 1,wherein the enzyme comprises a DNA or RNA polymerase enzyme.
 12. Thecircuit of claim 1, wherein the enzyme comprises E. coli DNA polymeraseI (Klenow) Fragment.
 13. The circuit of claim 1, wherein the enzymecomprises a genetically modified form of an E. coli Pol I polymerase, aBst polymerase, a Taq polymerase, a Phi29 polymerase, a T7 polymerase,or a reverse transcriptase.
 14. A method of sequencing a DNA or RNAmolecule, the method comprising: initiating at least one of a voltage ora current through a circuit comprising: a first electrode; a secondelectrode spaced apart from the first electrode; a first arm moleculehaving a first end and a second end, the second end of the first armmolecule electrically coupled to the first electrode; a second armmolecule having a first end and a second end, the second end of thesecond arm molecule electrically coupled to the second electrode; and anenzyme, wherein the first end of each of the first and second armmolecules are conjugated to two distinct sites on the enzyme so that aportion of the enzyme between the two distinct sites is included in thecircuit; exposing the circuit to a solution having an ionic strengthfrom dissolved ions and comprising dNTPs; and measuring electricalsignals through the circuit as the enzyme engages and processes a DNA orRNA template, wherein the electrical signals are processed to identifyfeatures that provide information on an underlying sequence of the DNAor RNA molecule when processed by the enzyme.
 15. The method of claim14, wherein the method provides information on an underlying sequence ofthe DNA molecule, and wherein the enzyme comprises a DNA polymerase. 16.The method of claim 14, wherein the method provides information on anunderlying sequence of the DNA molecule, and wherein the enzymecomprises a genetically modified form of an E. coli Pol I polymerase, aBst polymerase, a Taq polymerase, a Phi29 polymerase, or a T7polymerase.
 17. The method of claim 14, wherein the method providesinformation on an underlying sequence of the RNA molecule, and whereinthe enzyme comprises a reverse transcriptase.
 18. The method of claim14, wherein the method provides information on an underlying sequence ofthe RNA molecule, and wherein the enzyme comprises a Moloney murineleukemia virus reverse transcriptase, a porcine endogenous retrovirusreverse transcriptase, a bovine leukemia virus reverse transcriptase, amouse mammary tumor virus reverse transcriptase, a heterodimeric reversetranscriptase, or a Rous sarcoma virus reverse transcriptase.
 19. Themethod of claim 14, wherein the method provides information on anunderlying sequence of the RNA molecule, and wherein the enzymecomprises human immunodeficiency virus reverse transcriptase.
 20. Themethod of claim 14, wherein the electrical signals compriseperturbations in at least one of a current, voltage, impedance,conductivity, resistance or capacitance in the circuit.